Introduction. Yoghurts are traditionally made with cultures composed of Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus. However, in ...
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Selective enumeration of Lactobacillus casei in yoghurt-type fermented milks based on a 15°C incubation temperature Claude P. Champagne, Denis Roy and Alain Lafond Food Research and Development Centre, Agriculture and Agri-food Canada, 3600 Casavant Blvd., St-Hyacinthe Qué, J2S 8E3, Canada A procedure was developed to enumerate selectively Lactobacillus casei populations in yoghurt-type fermented milks that can also contain strains of Streptococcus thermophilus, Lactobacillus delbrueckii ssp. bulgaricus, Lactobacillus acidophilus and Bifidobacterium infantis. Commercial LBS agar was acidified to pH 5.4, and the plates were incubated at 15°C for 14 days under anaerobic conditions. Acidification prevented the development of streptococci, and incubation at 15°C limited the development of the lactobacilli and the bifidobacteria. L. casei formed colonies on HHD medium which were different from those obtained with L. bulgaricus. Counts of L. casei on HHD confirmed results obtained on LBS – pH 5.4 medium and incubated at 15°C. L. casei did not form colonies on M17, nor did L. acidophilus or L. bulgaricus.
Introduction Yoghurts are traditionally made with cultures composed of Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus. However, in the last decade, yoghurts have increasingly been enriched with various species of Bifidobacterium, cultures of Lactobacillus acidophilus and, more recently, Lactobacillus casei. This trend is a response by industry and consumers to data on potential probiotic properties of these species (McCann et al., 1996; Yaeshima, 1996). L. casei is the latest of the probiotic adjunct to be added to yoghurts or in probiotic cultures (McCann et al., 1996). In addition to its health potential, L. casei is of interest since it seems to be relatively stable during storage (Nighswonger et al., 1996), compared to the other yogurt cultures (Rhom et al., 1990; Rybka and Kailasapathy, 1995), and particularly bifidobacteria (Klaver et al., 1993; Shah et al., 1995). It is thus expected that an increasing proportion of fermented milks will contain L. casei. The bacterial flora of these new yoghurts is more diversified, thus increasing the challenge of following the evolution of the various populations during fermentation and storage. Media have been designed for the enumeration of bifidobacteria and L. acidophilus in fermented milks (Dave and Shaw, 1996; International Dairy Federation, 1995; Kneifel and Pacher, 1993; Onggo and Fleet, 1993; Rybka and Kailasapathy, 1996). However, there is little information on the selective enumeration of L. casei in yoghurts that may © 1997 Chapman & Hall
contain other lactobacilli such as L. bulgaricus and L. acidophilus, as well as bifidobacteria and streptococci. The substitution of glucose by melezitose in MRS agar has been used to enumerate L. casei from fermented products containing L. acidophilus and S. thermophilus (Valdez and Giori, 1993). The strategy based on the selective assimilation of carbohydrates was used in another study, where cellobiose was substituted to glucose in LBS agar (Nighswonger et al., 1996). This approach has the disadvantage of requiring the preparation of special media, and is potentially subject to variability in the carbohydrate assimilation patterns of strains. In this study, the ability of L. casei to grow at 15°C (Dellaglio et al., 1994) was used to develop a strategy of selective enumeration in fermented products having floras basically composed of thermophilic microorganisms. Lack of growth at 15°C is a recognized characteristic of L. acidophilus, S. thermophilus and L. bulgaricus (Dellaglio et al., 1994). The aim of this study was to establish the parameters of a plating procedure in which only colonies of L. casei would develop. Matereials and methods Bacteria The strains that were used as references for the validation of the methods were: Lactobacillus delbrueckii ssp. bulgaricus ATCC 11842, Lactobacillus acidophilus ATCC 4356, Lactobacillus casei ATCC 9595 and Bifidobacterium Biotechnology Techniques · Vol 11 · No 8 · 1997
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Table 1 Development and appearance of the reference strains on various culture media Media/temperature
B. infantis
L. bulgaricus
L. acidophilus
L. casei
CAB-raffinose/37°C M17/37°C LBS-5.4/15°C HHD/37°C
White colonies No growth No growth White colonies
No growth No growth No growth Blue colonies
No growth No growth No growth Blue colonies
No growth No growth White colonies Dark green colonies
infantis ATCC 27920. The lactobacilli were propagated on MRS broth (Difco), while the bifidobacteria were prepared on MRS broth supplemented with 0.5 g cysteine per L. Media Acidified LBS (LBS-5.4) was prepared by adjusting the pH of commercial LBS agar (Difco, Detroit) to 5.40 with glacial acetic acid. M17 agar with lactose was prepared as recommended by the manufacturer (Oxoid). The media were sterilized at 121°C for 15 min. The HHD agar medium was prepared as described by McDonald et al. (1987). The HHD agar base was composed of: 2.5 g fructose, 2.5 g KH2PO4, 10 g trypticase peptone, 1.5 g phytone peptone, 3.0 g casamino acids, 1.0 g yeast extract, 1.0 g Tween 80 and 20 g agar per L. The agar base was sterilized at 121°C for 15 min. A dye solution is prepared by dissolving 0.1 g of Bromcresol Green in 30 mL 0.01 M NaOH. Just before use, 2 mL of the dye solution is mixed with 100 mL the sterile molten (45–48°C) HHD agar base. Viable counts of bifidobacteria were obtained by plating on Columbia Agar Base (CAB; BBL Microbiology Systems, Cockeysville, MD) supplemented with 0.5 g cysteine, 5 g raffinose, 2 g LiCl and 3 g sodium propionate per L. The pH of the medium was adjusted to 5.1 with 6 M HCl and sterilized at 121°C for 15 min. Plates were incubated at 37°C for 48 h under anaerobic conditions (Roy et al., 1997). Analyses The reference strains and the commercial sample were analyzed by spreading 0.1 mL of the appropriate dilution on the surface of LBS-5.4, CAB-raffinose of HHD agars. All plates were incubated anaerobically. Those incubated at 37°C for 48 h were placed in a controlled atmosphere chamber having 5% CO2, 10% H2 and 85% N2, while a Gas Pack anaerobic jar system (BBL) was used for those incubated at 15°C for 14 days. Three different lots of a commercial yoghurt-type fermented milk (Danone Vitalité; Boucherville Qué), in which it was claimed that L. casei was included in the
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product, were analyzed to test the method in mixed culture conditions. Ten colonies obtained on LBS-5.4 were isolated and identified (Roy et al., 1997). Microscopic examination of the isolates was used to establish a cocci or rod cell form. Results and discussion Tests with pure cultures showed that the LBS-5.4/15°C method did not enable the development of the other lactobacilli and bifidobacteria used in the manufacture of yogurt-type products (Table 1). This suggested that the technique was appropriate, and an analysis of a commercial product was therefore carried out. Results of the analysis of the commercial products are shown in Table 2. The claims of the manufacturer, with respect to the bacterial content of its product, were confirmed. Microscopic examination of isolates of M17 agar showed that only the streptococci developed on this medium. It was known that M17 does not enable the development of L. bulgaricus colonies, but this study shows that the inhibitory activity of M17 towards lactobacilli extends to L. acidophilus and L. casei. Colonies of L. casei formed on HHD agar which enabled a confirmation of the data obtained on LBS-5.4. The L. casei colonies on HHD differed from those obtained with L. bulgaricus, which suggests that HHD could be used for differential enumeration of L. casei. Further data with more strains is warranted in this respect. Identification of the isolates from LBS-5.4 showed that only L. casei formed colonies on this medium following the 14 day incubation at 15°C. Selectivity between lactobacilli was related to the incubation temperature, since both L. acidophilus L. bulgaricus formed colonies on LBS-5.4 if incubated at 37°C.
Table 2 Bacterial population of Danone Vitalilé fermented milk. Data are average of three lots Medium
Species
Population (c.f.u./mL)
M17 HHD HHD and LBS-5.4
S. thermophilus L. bulgaricus L. casei
1.2 3 109 2.8 3 108 3.0 3 108
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Enumeration at 15°C can effectively select L. casei from the other yogurt cultures. The advantage of this approach is that it can be carried out with commercial media and does not require the preparation of specific media. In the case of the commercial product analyzed, which contained L. bulgaricus in addition to L. casei, LBS-5.4 medium could be used for the determination of the total lactobacilli count with an incubation at 37°C, which enables the determination of the L. bulgaricus by difference. One can expect that the procedure would also be adequate to acidified MRS agar, which is often used for the determination of lactobacilli in fermented milk products. If it is shown that lactococci do not form colonies on the acidified LBS agar, this approach also has the potential of analyzing L. casei populations in ripening Cheddar cheeses or fresh cheese, such as Quark, made with mesophilic cultures. Acknowledgements The technical assistance of Pierre Ward, Sylvie Landry and Daniel Vincent is gratefully acknowledged. This study was partially financed by Protégez-Vous. References Dave, R.I. and Shaw, N.P. (1996) J. Dairy Sci., 79: 1529–1536. Dellaglio, F., Roissard, H. de, Torriani, S., Curk, M.C. and Janssens, D. (1994) Caractéristiques générales des bactéries
lactiques. In: Les Bactéries Lactiques, Vol 1., pp 25–70, de Roissard H. & Luquet F.M. Eds., Lorica, Uriage. International Dairy Federation (1995) Detection and enumeration of Lactobacillus acidophilus. Culture Media. In: Bulletin of the IDF No 306, pp 23–33, IDF, ISSN 0250–5118, Brussels. Klaver, F.A.M., Kingma, F. and Weerkamp, A.H. (1993). Netherlands Milk Dairy J. 47: 151–164. Kneifel, W. and Pacher, B. (1993) Int. Dairy J., 3: 277–291. McCann, T., Egan, T. and Weber, G.H. (1996) J. Food Protection, 59: 41–45. McDonald, L.C., McFeeters, R.F., Daeschel, M.A. and Fleming, H.P. (1987) Appl. Environ. Microbiol., 53: 1382–1384. Nighswonger, B.D., Brashears, M.M. and Gilliland, S.E. (1996). J. Dairy Sci., 79: 212–219. Onggo, I. and Fleet, G.H. (1993) Austral. J. Dairy Technol., 48(2): 89–92. Rhom, H., Lechner, F. and Lehner, M. (1990) J. Food Protection, 53: 478–480. Roy, D., Mainville, I. and Mondou, F. (1997) Microecology and Therapy, in press. Rybka, S. and Kailasapathy, K. (1995) Austral. J. Dairy Technol., 50: 51–56. Rybka, S. and Kailasapathy, K. (1996) Int. Dairy J., 6: 839–850. Shah, N.P., Lankaputhra, W.E.V., Britz, M.L. and Kyle, W.S.A. (1995) Int. Dairy J., 5: 515–521. Valdez, G.F. de and Giori, G.S. de. (1993) J. Food Protection, 56: 320–322. Yaeshima, T. (1996) Benefits of bifidobacteria to human health. In: Bulletin of the IDF No 313, pp 36–41, International Dairy Federation, ISSN 0250–5118, Brussels.
Received 1 May 1997; Revisions requested 12 May 1997; Revisions received 2 June 1997; Accepted 5 June 1997
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